This is a competitive revision of the grant GM083849 "Microfluidics-based selections for the optimization of red fluorescent proteins." The broad goal of this project is to develop fluorescent proteins with an 80-fold improvement in signal output (e.g. number of photons emitted before photobleaching). Over the last 10-15 years, fluorescent proteins have provided critical insights into the fundamental workings of the cell as they enable researchers to visualize protein movements, enzyme activities, gene expression, and to quantify important signaling molecules in real time in living cells. As a result, fluorescent proteins have truly revolutionized cell biology, shedding light on the basic biology of cellular function, while helping to elucidate what goes wrong in disease states. Substantial improvements in the signal output of fluorescent proteins are required for the next level: visualizing and monitoring of single molecules within individual living cells. We hypothesize that an 80-fold improvement in signal output can be obtained by explicitly engineering both the chromophore pocket and surface environment of fluorescent proteins. This revision will add a new specific aim to the original three aims. The two main goals of the new aim are: i) to use biophysical data to guide library design of red fluorescent proteins (RFPs) and ii) to provide a mechanistic link between improved photophysical properties and changes in protein structure and dynamics. This new aim expands upon the original aims by using state-of-the-art nuclear magnetic resonance (NMR) techniques to collect atomic level structural and dynamic data for RFP mutants, which will accelerate the overall goal of developing improved fluorescent proteins. These data will be used to develop correlations between changes in structure and dynamics and photophysical properties (photostability, dark-state conversion, and brightness). We propose a combination of NMR methodologies to investigate peptide backbone and side chain structure and dynamics. This information will define regions of local conformational flexibility to be targeted in library designs. This will provide a more informed starting point for library design and streamline our efforts to generate improved fluorescent proteins and thus will accelerate the goals of the original proposal. PUBLIC HEALTH RELEVANCE: The development of new fluorescent proteins with substantial increases in signal output will dramatically expand our ability to visualize and probe the inner workings of living cells. These imaging tools will impact public health by providing valuable insight into basic biology and mechanisms of disease progression.